Lymphocytes are white blood cells which derive from either the thymus or the bone marrow. Lymphocytes cooperate in various ways to effect immunological responses. The bone marrow-derived or B lymphocyte cells (B-cells) secrete immunoglobulins in a primary or in a secondary immune response after appropriate antigen stimulation. Thymus-derived or T lymphocyte cells (T-cells) function as effector cells in cell mediated immune reactions, cooperate with B-cells to form immunoglobulin and suppress certain B-cell functions.
Monoclonal antibody technology first set forth by Kohler and Milstein (Nature 256:495-597 [1975]) allows production of reproducible monoclonal antibodies of predefined specificity. Lymphocytes now can be divided into subsets and further subpopulations by combining flow cytometry cell sorting techniques with specific membrane monoclonal antibodies directed to surface antigens. The state of maturation of the cell and functional differentiations based upon differing membrane surface structures, known as phenotyping, serve as population markers. Population marking with monoclonal antibodies enables study of the role of various populations of lymphocytes in regulating immune response and allows better recognition of the functions of the subpopulations. Functionally relevant cell surface molecules are identified by their ability to enhance or inhibit response to various stimuli.
Several in vitro tests are available to assess lymphocyte functions using various stimuli. In one such test, lymphocytes are activated with mitogens. For example, Phytohemagglutinin (PHA) or ConcanaviIin A (Con A) are mitogens which can be used to activate T-cells. Pokeweed Mitogen (PWM) can be used to stimulate B-cells. Also, antigen stimulation of leukocytes can be performed in culture by using various antigens, such as, Tetanus Toxoid (TT) and mumps. Other assessment methods available include mixed lymphocyte culture (MLC) and cell-mediated lympholysis.
Two major T-cell subsets are the CD4+ (T4+) subset and the CD8+ (T8+) subset. The CD4+ subset is involved in regulatory "helper-inducer" functions and antigen recognition functions wherein antigen or antigen-presenting cells are recognized in association with class II major histocompatability antigens (MHA-II). It also plays a major role in regulating immune response. E. L. Reinherz et al., Cell:19:821 (1980); C. Morimoto and Schlossman, S. F., Keio J. Med: 36:351 (1987). The CD8+ subset is involved with suppressor/cytotoxic functions and recognizes antigen in association with class I major histocompatability antigens (MHA-I). CD8+ subpopulations of cytotoxic effector and suppressor effector cells can be distinguished by using, for example, a monoclonal antibody such as the one described in our pending U.S. patent application Ser. No. 116,514, which enjoys common ownership.
Various CD4+ subsets of lymphocytes have been identified by monoclonal antibodies. Monoclonal antibody anti-Ta.sub.1 is expressed only on the T-cell lineage of cells and defines an activation antigen on T-cells. This monoclonal antibody immunoprecipitates a 105,000 dalton glycoprotein. D. A. Fox et al., J. Immunol. 133:1250 (1984). Anti-Ta.sub.1 identifies a minor subset of T-cells enriched for the anamnestic response to TT and mumps. Both Ta.sub.1 + and Ta.sub.1 - cells induce similar amounts of IgG synthesis by B-cells in the presence of PWM. D. A. Hafler et aI., J. ImmunoI. 137:414 (1986).
The anti-2H4 monoclonal antibody identifies the CD4+CD45RA+ suppressor-inducer cell population which is capable of triggering CD8+ suppressor-effector cells. C. Morimoto et al., J. Immuno. 134:1508 (1985); C. Morimoto et al., J. Immuno. 134:3762 (1985). In in vitro tests, this cell subset fails to respond to soluble antigens such as TT and mumps, and exhibits poor helper function in PWM and antigen driven antibody production systems. However, this cell subset proliferates maximally in the autologous mixed lymphocyte reaction (AMLR), a response of T-cells to selfclass II major histocompatibility complex (MHC) antigens. Anti-2H4 (CD45RA) monoclonal antibody defines the 200,000 and 220,000 dalton isoforms of the LCA/T200 family of antigens. C. E. Rudd et al., J. Exp. Med. 166:1758 (1987). These CD45R molecules are hypothesized to be involved in generation of suppressor-inducer activity. C. Morimoto et al., J. Immuno. 137:3247 (1986); T. Takeuchi et al., Eur. J. Immuno. 117:97 (1987); C. Morimoto et al., Eur. J. Immuno. 18:731 (1988).
Monoclonal antibody anti-4B4(CDw29) or anti-UCHL-1 (CD45RO) identifies the reciprocol CD4+CDw29+ subset of cells. S. H. Smith et aI., Immunology 58:63 (1986); M. Streuli et al., J. Immuno. 141:3910 (1988). The CD4+CDw29+ subset of cells thus responds maximally to recall antigen as memory T-cells and responds modestly in AMLR, but has poor suppressor-inducer activity. C. Morimoto et al., J. Immunol. 134:1508 (1985); C. Morimoto et al., J. Immunol. 134:3762 (1985). CDw29 molecules belong to the VLA/Integrin family of antigens which includes the fibronectin receptor and related structures. C. E. Rudd et al., J. Exp. Med. 166:1758 (1987); M. E. Hemler, Immuno. Today 9:109 (1988). This subset was determined to provide maximal help for B-cell immunoglobulin production. C. Morimoto et al., J. Immuno. 134:1508 (1985); C. Morimoto et al., J. Immunol. 134:3762 (1985). Also, the UHCL-1 and CDw29 antigens are proposed to be markers for memory T-cells. A. N. Abkar et al., J. Immuno. 140:2171 (1988); H. M. Serra et al., J. Immuno. 140:1435 (1988). Both UCHL-1 and CDw29 are on a high percentage of thymocytes (90% and 60%, respectively). However, this subset exhibits poor suppressor-inducer activity and responds poorly in AMLR. Both the CD4-CDw29. and CD4+CD45RA+ subsets of CD4+ cells react equally to alloantigen.
Other monoclonal antibodies capable of distinguishing T-cells and CD4+ subsets are known. These include anti-late differentiation antigen (anti-LDAl) (Suciu et al., Nature 318:465 [1985]), anti-D44 (A. Bernard et al., J. Immuno. 132:2338 [1984]; C. Calvo et al., J. Immuno. 136:1144 [1986]) and anti-Tp103 (B. Fleischer, J. Immuno. 138:1346 [1987]; B. Fleischer et al., J. Immuno. 141:1103 [1988]). However, none of these monoclonal antibodies can distinguish between two distinct functional populations, the population which responds maximally to the recall antigen and provides good helper function and the populations which cannot provide helper function or respond to the recallantigen in a population of CD4+ cells are precisely as the anti-lF7 monoclonal antibody.
Although the production of hybrid cell lines nad monoclonal antibodies is well known at this stage of implementation, great care must be exercised in the separation and maintenance of hybridoma cells insulture. Isolated clones have been known to produce antibodies against a subject antigen which differs from clone to clone since antibodies produced by different cells may react with different antigenic determinants on the same molecule. Adequate testing of the resulting antibody or antibody-containing medium, serum or ascitic fluid is essential. It is necessary to characterize the antibody of each clone which contributes to the complexity of producing monoclonal antibodies which are to be used in both diagnostic and therapeutic applications.
In developing a desied monoclonal antibody, one must identify and locate the antigenic determinant which will elicit a specific antibody to bind with it. Or, conversely, develop several hundred hybridoma clones from fusions performed and exhaustively screen them against normal and nonnormal tissue and different antigens to identify and define that clone which produces the antibody with the desired binding specificity. The object of this invention is to provide a monoclonal antibody which binds to a particular antigenic determinant expressed on the surface of human CD4+ (T4+) cells which enables such functional populations within this T-cell population to be determined. A monoclonal antibody is provided which enables a more precise phenotyping of the CD4+ population so as to distinguish between helper-inducer and suppressor-inducer populations. The CD4+lF7+ population responds maximally to recall antigen and provides good helper function, while the CD4+lF7- population cannot provide helper function or respond to the recall antigen. This monoclonal antibody also is preferentially expressed on the CD4+CDw29+ helper population, and thus can serve to subdivide further this population of cells.
The monoclonal antibody can be used in vitro to distinguish helper-inducer from suppressor-inducer CD4+ cells. The anti-lF7 monoclonal antibody can be labelled with a detectible compound so that immunological complexes can be detected. The label can comprise a dye, an enzyme, a fluorescent compound, a toxic reagent, a radioactive and electron dense element.
The monoclonal antibody also reacts with approximately 50% of CD8 cells and also reacts with B cells and macrophages. The function of such reaction of the monoclonal antibody has not been determined at this time.